TILLING-by-Sequencing+ Reveals the Role of Novel Fatty Acid Desaturases (GmFAD2-2s) in Increasing Soybean Seed Oleic Acid Content

Abstract

Soybean is the second largest source of oil worldwide. Developing soybean varieties with high levels of oleic acid is a primary goal of the soybean breeders and industry. Edible oils containing high level of oleic acid and low level of linoleic acid are considered with higher oxidative stability and can be used as a natural antioxidant in food stability. All developed high oleic acid soybeans carry two alleles; GmFAD2-1A and GmFAD2-1B. However, when planted in cold soil, a possible reduction in seed germination was reported when high seed oleic acid derived from GmFAD2-1 alleles were used. Besides the soybean fatty acid desaturase (GmFAD2-1) subfamily, the GmFAD2-2 subfamily is composed of five members, including GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E. Segmental duplication of GmFAD2-1A/GmFAD2-1B, GmFAD2-2A/GmFAD2-2C, GmFAD2-2A/GmFAD2-2D, and GmFAD2-2D/GmFAD2-2C have occurred about 10.65, 27.04, 100.81, and 106.55 Mya, respectively. Using TILLING-by-Sequencing+ technology, we successfully identified 12, 8, 10, 9, and 19 EMS mutants at the GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E genes, respectively. Functional analyses of newly identified mutants revealed unprecedented role of the five GmFAD2-2A, GmFAD2-2B, GmFAD2-2C, GmFAD2-2D, and GmFAD2-2E members in controlling the seed oleic acid content. Most importantly, unlike GmFAD2-1 members, subcellular localization revealed that members of the GmFAD2-2 subfamily showed a cytoplasmic localization, which may suggest the presence of an alternative fatty acid desaturase pathway in soybean for converting oleic acid content without substantially altering the traditional plastidial/ER fatty acid production.

Plant monounsaturated fatty acids: Diversity, biosynthesis, functions and uses

“…Aside from these breeding programs, biotechnological approaches have allowed the development of high-oleic oilseed crops by suppression of FAD2 gene(s) through targeted genome mutagenesis by means of TALENs [308], CRISPR/Cas9-mediated gene editing [309][310][311][312][313][314][315][316], or TILLING [317]. However, these techniques potentially impact fatty acid metabolism in all parts of the plant so that very high oleic acid phenotypes are usually associated with undesirable pleiotropic effects on crop performance [310,316].…”

The Soybean High Density ‘Forrest’ by ‘Williams 82’ SNP-Based Genetic Linkage Map Identifies QTL and Candidate Genes for Seed Isoflavone Content

“…For the Forrest cv., RNA-seq library was prepared by using four plant soybean tissues including seed, leaf, root, flower and pods as shown earlier [58]. From 100 mg of frozen grounded samples, total RNA was extracted using RNeasy QIAGEN Kit (Qiagen, Hilden, Germany).…”

“…[62]. Read count normalization and differential gene expression analysis were conducted using the Deseq2 package v1.30.1 [63] integrated in the OmicsBox platform from BioBam (Valencia, Spain) [58,64].…”

Direct access to millions of mutations by whole genome sequencing of an oilseed rape mutant population

“…(2017) demonstrated a TILLING by exome sequencing approach to detect EMS‐induced mutations in tetraploid and hexaploid wheat. The TbySeq approach has also been reported from several crops such as rice (Abe et al., 2012), maize (Nie et al., 2021), tomato (Garcia et al., 2016), soybean (Lakhssassi et al., 2021), sunflower (Fanelli et al., 2021) and cotton (Fang et al., 2020). In a first TILLING by whole genome sequencing (TbyWGS) approach for oilseed rape, a limited number of EMS mutants were whole genome sequenced to detect EMS‐induced mutations (Tang et al., 2020).…”